Resolvin D3 and Aspirin-Triggered Resolvin D3 Are Protective for Injured Epithelia

Acid-Induced Lung Injury in Mice

All animal protocols were reviewed and approved by the Harvard Medical School standing committee on animals (protocol 03618). Mice were housed in an American Association for Laboratory Animal Science–accredited facility. As described previously,¹³ male BALB/c mice (6 to 10 weeks; Charles River, Wilmington, MA) were anesthetized with ketamine/xylazine, and 50 μL of hydrochloric acid (pH 1.0, 0.1N, 0.22 μm filter-sterilized; Sigma-Aldrich, St. Louis, MO) was instilled selectively into the left mainstem bronchus. Some mice received AT-RvD3 (10 ng/mouse) or vehicle control [0.1% (v/v) ethanol] in saline via i.v. injection 1 hour after injury was induced. Mice were euthanized at 6, 24, 48, or 72 hours after injury. In a separate experiment, mice received AT-RvD3 (10 ng, i.v.) or vehicle [0.1% (v/v) ethanol] at 24 and 48 hours, and euthanized 72 hours after intratracheal HCl. To ensure molecular integrity of AT-RvD3 stock and determine concentration, the compound was assessed by UV-visible light (UV-vis) spectrophotometry before preparation of the working solution. A representative spectrogram of AT-RvD3 is shown in Supplemental Figure S1.

Identification of Endogenous RvD3 in Murine Lung

BALB/c mice were subjected to the acid-injury protocol, as described above, and allowed to recover for 24 to 72 hours. Briefly, lungs from acid-injured mice and uninjured controls were removed, gently homogenized in 1 mL ice-cold methanol, and lipid mediators were extracted, as described before,12, 14 using C18 columns for liquid chromatography tandem mass spectrometry analysis. RvD3 and the pathway markers 4- and 17-hydroxydocosahexaenoic acid (HDHA) were monitored and quantified by multiple reaction monitoring using diagnostic ion fragmentation patterns.

Histology and Immunohistochemistry

Lungs were perfusion fixed with zinc fixative (BD Pharmingen, San Diego, CA) at 20 cm H₂O, as done previously.⁸ Tissues were embedded, divided into sections, and stained with hematoxylin and eosin by either the Dana Farber/Harvard Cancer Center Rodent Histopathology Core or the Brigham and Women's Pulmonary and Critical Care Medicine Histology Core. Tissue sections were deparaffinized and rehydrated in graded ethanols and water; a subset of slides was stained with hematoxylin and eosin following standard protocols. For immunohistochemistry using antibodies for pNF-κB and Ki-67, antigen retrieval was performed by boiling slides in 10 mmol/L citrate buffer, pH 6; antigen retrieval was not performed for lymphatic vessel endothelial hyaluronan receptor (LYVE) 1 and vascular endothelial growth factor receptor (VEGFR) 3 antibodies. Slides were blocked with 10% normal goat serum diluted in 1% bovine serum albumin dissolved in Tris-buffered saline with 0.025% Tween-20 for 1 hour, followed by avidin-biotin blocking (Invitrogen, Grand Island, NY). Primary antibodies were diluted in 1% bovine serum albumin dissolved in Tris-buffered saline with 0.025% Tween-20; samples were incubated at 4°C overnight: pNF-κB (S536) (Cell Signaling, Danvers, MA; 1:100), Ki-67 (Abcam, Cambridge, MA; 1:1000), LYVE1 (AngioBio, DelMar, CA; 1:10,000), VEGFR3 (R&D Systems, Minneapolis, MN; 1:2000). The secondary antibody used was goat anti-rabbit (Vector Laboratories, Burlingame, CA; 1:5000, 2 hours, room temperature). Slides were developed using a 3′,3′-diaminobenzidine kit (Invitrogen). Negative controls for immunohistochemistry consisted of incubating slides in the absence of primary antibody.

Enumeration of Ki-67⁺ Epithelial Cells and LYVE1⁺/VEGFR3⁺ Lymphatic Vessels

To quantitate proliferation in mouse lung epithelia, images of five distinct fields per injured lung were taken at ×400 magnification. Total and Ki-67⁺ cells were counted for each field; results are broken down for alveolar and airway epithelial compartments.

For identification and enumeration of LYVE1⁺VEGFR3⁺ lymphatic vascular structures, stained slides were scanned at the Dana Farber/Harvard Cancer Center Tissue Microarray and Imaging Core using an Aperio slide scanning device (Leica Biosystems, Inc., Buffalo Grove IL). Scanned slides were viewed using Aperio's ImageScope software version 12.1.0.5029 (Leica Biosystems, Inc.); lymphatic vessels were identified by LYVE1 and VEGFR3 positivity. Total numbers of LYVE1⁺ and VEGFR3⁺ vessels were counted in the left (injured) lung for each animal.

Lung Fluid Clearance

To determine differences in total lung water levels, we performed wet/dry weight ratio analyses. Lungs were removed at necropsy and weighed immediately. Lungs were dried for 48 hours at 55°C and reweighed. The wet/dry weight ratio was calculated by dividing the initial wet weight by the dry weight.

Western Blot Analysis

Whole lung lysates were prepared by homogenizing tissues in cold radioimmunoprecipitation assay buffer supplemented with Complete mini protease inhibitor cocktail and PhosSTOP phosphatase inhibitors (Sigma-Aldrich). Protein concentrations were measured using a BCA protein assay kit (Thermo Fisher, Cambridge, MA). Any kD precast gels were loaded with 40 μg/well protein and run at 100 V (Bio-Rad, Hercules, CA). Proteins were transferred to polyvinyl difluoride membranes overnight (4°C, 25 V). For detection of selected proteins, blots were blocked for 1 hour with 5% skim milk in Tris-buffered saline with 0.025% Tween-20. Primary antibodies were diluted in 5% milk–Tris-buffered saline with 0.025% Tween-20 buffer. We evaluated expression of surfactant proteins D and A (SP-D and SP-A) in bronchoalveolar lavage fluid (BALF); for these blots, 30 μL of BALF was loaded per well. Primary antibody sources and concentrations were as follows: phospho-NF-κB (S536) (Cell Signaling), 1:1000; NF-κB (BioLegend, San Diego, CA), 1:1000; epithelial sodium channel (ENaC) γ (Abcam), 1:1000; aquaporin 5 (Millipore, Billerica, MA), 1:1000; and α-tubulin (Abcam), 1:5000. Antibodies for SP-D and SP-A were a gift from Dr. David Douda (Brigham and Women's Hospital/Harvard Medical School), Dr. Nades Palaniyar, and Pascal Dijadeu (Hospital for Sick Children, Toronto, ON, Canada; 1:10,000). Blots for all antibodies except SP-D and SP-A were incubated overnight at 4°C; SP-D and SP-A blots were incubated with primary antibody for 2 hours at room temperature. A horseradish peroxidase–conjugated goat anti-rabbit antibody (1:10,000, 2 hours at 25°C) was used as a secondary antibody (Santa Cruz Biotechnology, Dallas, TX). Tissue lysates suggested in the antibody data sheets as positive or negative for specific proteins were used as controls. Chemiluminescence was developed using the Pierce Supersignal Pico Chemiluminescence substrate kit (Thermo Fisher, Cambridge, MA). Images were taken using the ChemiDoc XRS system (Bio-Rad). Densitometry was performed using ImageJ software version 1.49t (NIH, Bethesda, MD).

Growth Factor and Cytokine Analysis

IL-6 levels were measured using a LegendPlex Mouse Inflammation kit, following manufacturer's instructions (BioLegend). Keratinocyte growth factor (KGF; alias FGF7) was measured in BALF by enzyme-linked immunosorbent assay, per manufacturer's instructions (RayBiotech, Norcross, GA).

Bronchoalveolar Lavage Collection and Cytospin Preparation

Bilateral BAL was performed with two aliquots of phosphate-buffered saline + 0.6 mmol/L EDTA (24-hour time point). Total cell counts and leukocyte differentials were performed as described.¹⁵ Briefly, BALFs were spun down, and the supernatant collected for analyses of soluble mediators. The cell pellet was subjected to red cell lysis. Total cell counts were performed using a hemacytometer. A fraction of the BALF cells was used for cytospins; leukocyte differentials were performed after staining cytospin preparations with Wright-Giemsa (Sigma-Aldrich).

Cell Culture Experiments

E. coli Pneumonia

Mice were prepared for intratracheal instillation of live bacteria to the left lung, as described above and previously.¹⁹ A total of 1 × 10⁶ colony-forming units of Escherichia coli were introduced to the left mainstem bronchus (total volume, 25 μL). Mice were given AT-RvD3 (10 ng, i.v.) 1 hour after surgery. Mice were allowed to recover from anesthesia and returned to their cages. At 6 hours after surgery, mice were euthanized; lungs were harvested aseptically and homogenized in ice-cold sterile saline. To determine colony-forming units (CFUs), lungs were homogenized and serial dilutions were plated on Lysogeny broth agar plates. Colonies were counted after 24 hours' incubation at 37°C. Results are expressed as CFU per whole lung. Bacterial growth index was calculated as the ratio of lung CFU/CFU in the original inoculum.¹⁹

Statistical Analysis

Data analysis was performed using GraphPad Prism software version 5.01 (GraphPad Software, Inc., La Jolla, CA). Data are expressed as means ± SEM unless stated otherwise. Results were considered statistically significant if P ≤ 0.05. Comparisons were performed using t-test (for larger sample sizes with normal distribution) or Mann-Whitney U test (smaller sample sizes and/or nonnormally distributed data) unless otherwise noted. See Results and figure legends for details of specific tests performed.

Endogenous RvD3 Is Generated in Response to Acid-Induced Lung Injury

To determine whether RvD3 was generated after acid-initiated injury of murine lungs, we performed lipidomics analysis on lung extracts (described in Materials and Methods). RvD3 and its biosynthetic pathway intermediates 4-HDHA and 17-HDHA were identified in accordance with published criteria,¹⁴ including retention time and MS-MS fragmentation spectra (Figure 1, A and B). Relative to uninjured lungs, 4-HDHA and 17-HDHA increased approximately twofold to threefold 24 hours after injury, returning to baseline levels at 72 hours (Figure 1C). RvD3 was present in uninjured lungs (26.3 ± 7.8 pg/lung) and significantly increased by approximately twofold at 24 hours (56.7 ± 5.8 pg/lung), and threefold by 72 hours (80.7 ± 15.7 pg/lung) in injured lungs (Figure 1D).

AT-RvD3 Limits Acute Lung Injury

Because of its increased resistance to enzymatic degradation, we used AT-RvD3, a 17R-epimer of RvD3, for in vivo experiments.12, 20 Lung histology revealed interstitial and alveolar edema and inflammation in the left lung of acid-injured animals that was maximal at 24 hours and slowly decreased at 48 and 72 hours (Figure 2, A–D and F–H). Relative to vehicle alone (Figure 2, A–D), mice that received AT-RvD3 (10 ng/mouse, i.v., 1 hour after injury) had decreased histopathological correlates of ALI (n ≥ 3) (Figure 2, E–H). In mice treated with AT-RvD3, there were reductions in edema, alveolar wall thickening, inflammatory cell infiltrates, and hemorrhage (Figure 2, E–H). Higher-magnification images are shown for the 24-hour time point (Supplemental Figure S2).

AT-RvD3 Enhances Resolution of Edema

In view of the histological changes in lung edema with AT-RvD3, lung wet/dry weight ratios were determined 24 hours after acid instillation. AT-RvD3 administration led to a significant reduction in lung water (Figure 3A). Because ENaC serves a major role in alveolar water clearance,21, 22 we next measured whole lung expression of the ENaCγ subunit (Figure 3B). Results were quantitated by densitometry and normalized to α-tubulin. Levels of ENaCγ were decreased from baseline (data not shown) in vehicle-treated mice; ENaCγ expression was significantly increased in the presence of AT-RvD3 (Figure 3C). We also measured expression of the water channel protein aquaporin 5 at 24 hours after injury by Western blot analysis; no significant difference was observed between the groups (Supplemental Figure S3).

Because lymphatics serve an important function in the clearance of lung tissue edema,²³ we determined if AT-RvD3 influenced the number of detectable lymphatic vessels. To visualize the lymphatics, lung tissue was stained for expression of LYVE1 (Figure 3, D and E) and VEGFR3 (Supplemental Figure S4). Seventy-two hours after injury, there were increased numbers of LYVE1⁺ lymphatic vessels detected in mice treated with AT-RvD3 relative to controls (Figure 3D). The lymphatics were enumerated and approximately 50% more LYVE1⁺ vessels were present with AT-RvD3 relative to vehicle (P < 0.02, n ≥ 4 per group, Mann-Whitney U test) (Figure 3E). VEGFR3⁺ vessels increased with AT-RvD3, but did not reach statistical significance with this sample size (n = 3) (Supplemental Figure S4).

AT-RvD3 Limits Leukocyte Infiltration to Acid-Injured Lung

To examine the impact of AT-RvD3 on tissue inflammation, lung leukocyte subsets were enumerated in BALFs of mice 6, 24, and 72 hours after initiation of injury; mice were given AT-RvD3 or vehicle 1 hour after injury (described in Materials and Methods) (Figure 4A). At 6 hours after injury, AT-RvD3 led to an approximately threefold reduction in the number of neutrophils [171 ± 17.13 (vehicle); 54.53 ± 11.38 (AT-RvD3)] (Figure 4B). Macrophages were reduced almost twofold in AT-RvD3 treated mice at this time point [146 ± 23 (vehicle); 74 ± 8.63 (AT-RvD3)] (Figure 4B). At 24 hours after injury, AT-RvD3 led to a dramatic and statistically significant reduction in BALF neutrophils [0.99 × 10⁵ ± 0.19 × 10⁵ (vehicle) and 0.19 × 10⁵ ± 0.06 × 10⁵ (AT-RvD3)] (Figure 4C). At 24 hours, no significant difference in total numbers of BALF macrophages was evident with AT-RvD3 (Figure 4C). A statistically significant decrease in total lymphocytes was found with AT-RvD3 treatment [2.70 × 10³ ± 0.37 × 10³ (vehicle) versus 1.60 × 10³ ± 0.32 × 10³ (AT-RvD3); P < 0.04, n ≥ 14 mice per group] (data not shown). Seventy-two hours after injury, neutrophils are significantly reduced in the AT-RvD3–treated mice [0.70 × 10⁵ ± 0.15 × 10⁵ (AT-RvD3), 1.64 × 10⁵ ± 0.32 × 10⁵ (vehicle)] (Figure 4D); no significant difference was observed in the number of macrophages (Figure 4D).

To determine the impact of administration of AT-RvD3 at later time points, mice were subjected to acid injury as described, and received doses of AT-RvD3 (10 ng/mouse, i.v.) at 24 and 48 hours after injury (Figure 4E). Total leukocytes were significantly increased in AT-RvD3–treated mice relative to vehicle (n = 4 per group; P = 0.028) (Figure 4F). Mice receiving AT-RvD3 had fewer neutrophils (0.89 × 10⁵ ± 0.23 × 10⁵) relative to controls (4.41 × 10⁵ ± 0.89 × 10⁵) (Figure 4G). Macrophage numbers were not significantly different between the groups (Figure 4H).

RvD3 and AT-RvD3 Limit Activation of the NF-κB Pathway

Lung injury elicits NF-κB activation as a key regulator of tissue inflammation,²⁴ so we next explored the influence of AT-RvD3 on NF-κB expression and activation. First, in vitro, we used a lung epithelial cell line stably transfected with a fluorescent NF-κB reporter (described in Materials and Methods). Tumor necrosis factor-α gave a robust, dose-dependent increase in fluorescence (Figure 5, A and B). RvD3 and AT-RvD3 (10 nmol/L) significantly inhibited tumor necrosis factor-α–induced NF-κB activation (Figure 5, A and B). To detect NF-κB activation in vivo, we used a phospho-NF-κB (pNF-κB) antibody for serine 536 for Western blot analysis and immunohistochemistry (Figure 5, C–E). At 24 hours after injury, lung pNF-κB was detected by Western blot analysis (Figure 5C). Mice receiving AT-RvD3 had consistently decreased activation by approximately 1.5-fold (Figure 5D). To identify cellular sources of pNF-κB, we performed immunohistochemistry with the same antibody used for Western blot analysis. NF-κB activation was apparent in both epithelial cells and leukocytes, and was decreased with AT-RvD3 (Figure 5E). As a marker of functional regulation of NF-κB,²⁵ BALF levels of IL-6 were determined. In response to AT-RvD3, levels of IL-6 were significantly reduced 24 hours after injury (Figure 5F).

AT-RvD3 Promotes Increased Proliferation of Epithelial Cells after Acid Injury

After acid injury, epithelial cell proliferation is required for restitution of the mucosal barrier. Airway epithelial cells stained positive for the nuclear protein Ki-67 24 hours after injury (Figure 6A). The percentage of Ki-67⁺ cells by immunohistochemistry was increased by AT-RvD3 in both airway and alveolar compartments (Figure 6, A and B). To quantitate the direct actions of AT-RvD3 on epithelial responses to injury, we used the Electrical Cell-Substrate Impedance Sensing in vitro assay with Calu3 cells that (unlike A549 cells) form a tight barrier (described in Materials and Methods). After induction of injury, Calu3 cells exposed to 10 nmol/L AT-RvD3 displayed an accelerated increase in restitution of an intact epithelial barrier; statistically significant differences between treatment groups were apparent as early as 10 hours after injury (Figure 6C).

AT-RvD3 Enhances Reparative Responses and Re-Epithelialization in Models of Lung Injury and Skin Wounding

KGF has been linked to epithelial restitution in both skin and lung,26, 27 so the influence of AT-RvD3 on BALF and whole lung lysate levels of this cytokine was next determined. AT-RvD3 led to significant increases in BALF KGF [116.4 ± 31.1 pg KGF/μg protein (AT-RvD3) versus 47.7 ± 7.0 pg KGF/μg protein (vehicle)] (Figure 7A). Similarly, AT-RvD3 treatment led to increased KGF in samples of whole lung lysates [234.4 ± 9.05 (vehicle) versus 295.4 ± 20.84 (AT-RvD3)] (Figure 7B). Messenger RNA for the KGF receptor Fgfr2 was also expressed in lung 24 hours after injury [mean ΔC_t ± SEM: 4.29 ± 0.22 (AT-RvD3) and 4.46 ± 0.21 (vehicle)]. As KGF is also known to affect surfactant protein levels in response to lung injury, we measured levels of surfactant proteins A and D (SP-A and SP-D) in BALF from mice 24 hours after acid injury. At this time point, we did not observe any clear difference between vehicle and AT-RvD3 treated groups (Supplemental Figure S5).

Because KGF serves important roles in skin wound healing,²⁷ we next determined whether epithelial responses to AT-RvD3 were exclusive to the lung or represented a more general action in promoting resolution. We used a model of full-thickness skin wounding that is characterized by initial re-epithelialization of the defect (described in Materials and Methods). AT-RvD3 (100 ng administered topically, once a day for 5 days) led to more rapid re-epithelialization than vehicle, as determined by visual inspection (Figure 7, C and D) and tissue histology (Figure 7E). The degree of re-epithelialization was significantly greater by day 3 after injury in mice given AT-RvD3 (Figure 7D). Representative histology revealed a more rapid restoration of normal, full-thickness epidermis in mice receiving AT-RvD3 (Figure 7E).

To determine the impact of AT-RvD3 on bacterial clearance, we subjected a group of mice to the E. coli pneumonia protocol (described in Materials and Methods). AT-RvD3 (10 ng, i.v.) did not lead to a worsening of infection, as assessed by measurement of CFU and bacterial growth index at 6 hours after instillation of bacteria (Supplemental Figure S6).